水稻花器官数目异常突变体afon1的表型分析与基因定位

发布时间：2018-05-24 01:12

本文选题：水稻 + 花器官数目异常突变体(afon)　；参考：《中国农业科学》2017年20期

【摘要】：【目的】研究水稻花器官数目异常突变体afon1(abnormal floral organ number1)的分子机理,鉴定出控制水稻花器官数目变化的基因。【方法】利用甲基磺酸乙酯(EMS)诱变籼稻品种浙农34获得一个花器官数目异常突变体作为试验材料,命名为afon1。开花期随机取突变体afon1和野生型浙农34的稻穗各5个,利用组织学和扫描电子显微镜等技术研究afon1的花器官表型、细胞学特征和花粉育性。成熟期随机取突变体afon1和野生型浙农34植株各10株,测定株高、分蘖数、穗长、每穗颖花数、每穗实粒数和千粒重等农艺性状。随机取突变体afon1和野生型饱满种子各100粒,测定发芽势和发芽率。以突变体afon1为母本,分别与野生型浙农34和粳稻品种浙农大104杂交构建2个F2群体进行遗传分析和基因定位,筛选候选基因进行DNA测序比对,构建AFON1蛋白质的空间模型并对其结构进行分析,同时对候选基因以及与花器官数目相关的基因进行实时荧光定量PCR分析。【结果】与野生型相比,突变体afon1中59.64%小穗的花器官数目发生异常,其中多数小穗仅在内稃一侧产生一个颖壳状的器官,部分小穗表现2—4轮花器官数目同时增加;株高和千粒重显著增加,而结实率显著降低。遗传分析表明,突变体afon1与野生型浙农34杂交的F1植株小穗花器官数目表现正常,F2群体中小穗花器官数目正常植株与花器官数目异常植株的分离比符合3㑳1,表明突变体afon1性状受一对隐性核基因控制,基因位于水稻第1染色体长臂端In Del标记1M5和1M18之间,物理距离为73 kb,该区间内共有6个注释基因。突变体afon1和野生型的测序比对发现,突变体afon1中的基因LOC_Os01g67430外显子中第565个碱基T突变成A,导致第189个氨基酸由色氨酸突变为精氨酸。蛋白质序列和空间结构分析表明,AFON1蛋白质序列中含有一个Lipase_3结构域,结构域内的突变导致蛋白质的空间结构发生了明显的变化。实时荧光定量PCR结果显示,LOC_Os01g67430在突变体afon1幼穗中的表达量要显著高于野生型,而在根、茎和叶中则无显著差异;穗发育早期FON1和FON2/4等调控花器官数目的基因在突变体afon1花器官中的表达量显著增加。【结论】LOC_Os01g67430为突变基因afon1,该基因通过影响花器官数目相关基因的表达而调控各轮花器官数目。
[Abstract]:[objective] to study the molecular mechanism of afon1(abnormal floral organ number1, a mutant with abnormal floral organ number in rice. [methods] A mutant with abnormal floral organ number was obtained by mutating indica rice variety Zhennong 34 with ethyl methanesulfonate EMS. as the experimental material, named afon1. The floral organ phenotype, cytological characteristics and pollen fertility of afon1 were studied by means of histology and scanning electron microscopy (SEM). At maturity stage, 10 plants of afon1 and 10 plants of wild type Zhejiang nong 34 were randomly selected. Agronomic characters such as plant height, tiller number, ear length, spikelet number per panicle, grain number per panicle and 1000-grain weight were measured. The germinating potential and germination rate of the mutant afon1 and wild type full seeds were determined. Two F2 populations were constructed by hybridization with wild type Zhejiang nong 34 and japonica rice variety Zhejiang Nongda 104 using afon1 as female parent. The candidate genes were screened for DNA sequencing. The spatial model of AFON1 protein was constructed and its structure was analyzed. Meanwhile, the candidate genes and genes related to the number of floral organs were analyzed by real-time quantitative PCR. [results] compared with wild type, In the mutant afon1, 59.64% of spikelet floral organs were abnormal, and most spikelets produced only one glume in one side of Palea, some spikelets showed 2-4 floral organs increasing simultaneously, plant height and 1000-grain weight increased significantly. The seed setting rate decreased significantly. Genetic analysis shows that The number of small spikelet organs of F1 plants crossed by afon1 and wild type Zhejiang nong 34 showed that the segregation ratio of normal spikelet organ number to abnormal floral organ number in F _ 2 population was in accordance with 3Y1, which indicated that the mutant afon1 was affected by one plant. Control of recessive nuclear genes, The gene is located between in Del labeled 1M5 and 1M18 on rice chromosome 1, and the physical distance is 73 kb. There are 6 annotated genes in this region. Sequencing of the mutant afon1 and wild-type showed that the 565th base T of the gene LOC_Os01g67430 exon of the mutant afon1 was mutated to A, resulting in the 189th amino acid mutation from tryptophan to arginine. The analysis of protein sequence and spatial structure showed that AFON1 protein sequence contained a Lipase_3 domain, and mutations in the domain resulted in obvious changes in the spatial structure of the protein. The results of real-time fluorescence quantitative PCR showed that the expression of Os01g67430 in young spike of mutant afon1 was significantly higher than that in wild type, but there was no significant difference in root, stem and leaf. In the early stage of panicle development, the expression of FON1 and FON2/4 genes regulating the number of floral organs increased significantly in the floral organs of mutant afon1. [conclusion] LOC_Os01g67430 is a mutant gene afon1, which is regulated by affecting the expression of floral organ number related genes. Number of flower organs per rosette.
【作者单位】：浙江大学农业与生物技术学院农学系;
【基金】：浙江省重大科技攻关专项(2012C12901-2) 浙江省科技厅公益技术应用研究计划(2016C32085) 高等学校学科创新引智计划(Grant B14027) 教育部创新团队资助项目(IRT1185)
【分类号】：S511

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